In the C3H/10T1/2 CL8 cell line we have demonstrated that the expression of cells malignantly transformed by chemical carcinogens can be repressed by contact of malignant cells with nontransformed cells and that this inhibition can be modulated by serum concentration and by inhibitors of phosphodiesterase. It is the major aim of this research to examine in greater detail correlations between alterations in cyclic nucleotide levels and alterations in growth inhibition and to examine how this intracellular communication occurs in vitro. A good correlation has been achieved between elevations of intracellular cAMP levels and growth inhibition of malignant cells in mixed cultures, and these cells have been shown to be in metabolic communication. Using mixed culture of carcinogen-initiated cells and parental 10T1/2 cells, the colony size attained by these initiated cells was shown to directly determine the probability for transformation of these cells, indicating the central role played by cell interactions in expression of malignancy. We are currently studying the extent of intracellular communication using dye-injection procedures and examining modifications induced by elevations of cAMP levels, and how such communication differs among a series of transformed lines selected to be sensitive or resistant to growth inhibition. The role of cAMP will also be probed using kinase-deficient cell mutants. The relevance of the observed in vitro effects to the in vivo situation will be investigated by studying the growth rate of transplantable tumors, of differing sensitivity to growth inhibition in vitro, in host animals treated with drugs which modify the in vitro response. We will also extend these studies to human fibroblasts in culture which we have shown to secrete an inhibitory factor into the medium. We will characterize this factor biologically and biochemically. These studies, in addi- tion to providing basic information on growth control mechanisms, will aid in the development of more reliable and quantitative in vitro assays for chemically induced malignant transformation in mouse and human cell lines. (A)